<p> <i>Ustilago maydis</i> is a basidiomycete fungus known to cause tumor formation in maize. It exhibits two distinct forms: yeast-like and filamentous. Both forms exhibit cell polarity, an inherent process observed in all cell types. In the Ascomycota, Tea1 homologs are involved in cell morphogenesis. Tea1 homologs had not been identified in the Basidiomycota. In this study, I have characterized the homolog of the <i>Schizosaccharomyces pombe tea1</i> gene in <i>U. maydis</i>. I have determined the &Delta;<i> tea1</i> null phenotype through a gene deletion strategy, established the subcellular localization of Tea1 and Tea4 using GFP fusions, and determined a Tea1 interaction with itself using a yeast two-hybrid approach. These studies provide support for a major role of Tea1 in cell morphogenesis, in maintaining an axis of polarized growth, and in cell polarity in <i>U. maydis</i>. </p>

<p> In the presence of arachidonic acid (AA), <i>Saccharomyces cerevisiae </i> produces prostaglandin E₂ (PGE₂). <i> S. cerevisiae</i> and its metabolites may be consumed in products manufactured using the yeast (e.g. beer). Neutrophils are immune cells present in the gastrointestinal (GI) tract during inflammation. As a lipid-signaling molecule, PGE₂ can potentially modify neutrophil functions and exacerbate pre-existing inflammation. As neutrophil migration is a hallmark of inflammation, we investigated the impact of PGE₂ on neutrophil chemotaxis. Chemotaxis assays were performed on neutrophils isolated from human whole blood using the chemotactic agents f-Met-Leu-Phe (fMLP) or interleukin-8 (IL-8). Neutrophil chemotaxis was concentration dependent as it was enhanced 3.5-fold at low concentrations of PGE₂ (0.1 nM-10 nM) and reduced 3.0-fold at higher concentrations of PGE₂ (100 nM).</p><p> The biochemical pathway utilized by <i>S. cerevisiae</i> to produce PGE₂ is unknown. Identifying enzymes that metabolize AA may direct approaches to reduce the impact that yeast PGE₂ may have on neutrophils. <i>S. cerevisiae</i> does not have genes homologous to those involved in mammalian AA metabolism. We employed RNAseq transcriptome sequencing to study the lipid biosynthetic pathway in <i>S. cerevisiae </i> and observed 1248 genes upregulated in yeast that were cultured in the presence of AA relative to yeast that were cultured without AA. Notably, genes that mediate beta-oxidation of fatty acids (<i>Pot1, Pox1, Faa1 and Faa2</i>) were upregulated up to 2.3-fold.</p><p> The results demonstrate that low concentrations of PGE₂ enhance neutrophil chemotaxis that is mediated by fMLP or IL-8, suggesting that PGE₂ may aid in recruiting neutrophils from regions that are distant to a site of inflammation. Once a higher concentration of PGE₂ is encountered by neutrophils, neutrophils may halt their migration and engage effector functions such as phagocytosis and superoxide production. Increased expression of genes involved with fatty acid metabolism points to enzymes that may utilize AA to produce PGE₂ in <i>S. cerevisiae</i>. Experiments testing PGE₂ levels in knock-out strains of yeast will identify genes involved in PGE₂ production. Results of this study have implications to reduce potential off-target effects caused by yeast PGE₂ in consumables.</p>

<p> Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in <i>Vibrio cholerae,</i> we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in <i> V. cholerae,</i> ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT <i>in vitro</i> binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the <i>Vibrio</i> Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated <i>tarB</i>, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of <i>V. cholerae</i> infection. Infections progressing for 18 hours or less showed the &Delta;<i>tarB</i> strain was out-competed by the wild type strain, while those carried out longer, showed &Delta;<i> tarB</i> out-competing the wild type. Additionally, if inoculated from a resource poor environment the &Delta;<i>tarB</i> strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that <i>tarB</i>-mediated regulation of the gene <i> tcpF</i> was primarily responsible for the <i>tarB</i> mutant's <i> in vivo</i> colonization phenotype. Further investigation of genes regulated by <i>tarB</i> using genome-wide transcriptional profiling of a <i> tarB</i> over-expressing strain revealed that <i>tarB</i> also directly regulates genes involved in iron and amino acid uptake. We determined that <i>tarB</i> has a repressive effect on many genes within the VPI-1, but has an activating effect on <i>tcpP/tcpH,</i> encoding regulators upstream of ToxT. Taken together, the data suggest that <i> tarB</i> plays an important role in regulating virulence and metabolic genes early after <i>V. cholerae</i> infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication <i> in vivo.</i></p>

<p> Doc induces cell death by inhibiting translation; however, the mechanism of Doc-induced cell death and the cellular target of the toxin were unknown. One theory suggested that Doc inhibits translation elongation by binding directly to the 30S ribosomal subunit. Later evidence showed catalytic activity in distant homologs of Doc. These homologs contain a Fic-domain that has been shown to modify target GTPases by AMPylation and phosphocholination. Therefore, [³⁵S] &ndash; Met, &alpha;[³²P] &ndash; ATP, and &gamma;[³²P] &ndash; ATP were used in conjunction with an S30 extract to confirm that Doc inhibits translation, to assess the mechanism of modification, and to identify the modified target. The results showed that Doc is an enzyme that inhibits translation and phosphorylates a protein target.</p>

Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5726. Adviser: Yves V. Brun. Title from dissertation home page (viewed May 12, 2008).

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7106. Adviser: William Metcalf. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2007. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0822. Adviser: Gary Olsen. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-04, Section: B, page: 2065. Adviser: Jeffery Cox.

Thesis (Ph.D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6419. Adviser: Tristram Parslow.

Thesis (Ph.D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 69-09, Section: B, page: 5220. Adviser: Joseph L. DeRisi.